METHODS FOR GENERATING AMPLIFIED NUCLEIC ACID ARRAYS

• US 20080242560A1
• Filed: 11/20/2007
• Published: 10/02/2008
• Est. Priority Date: 11/21/2006
• Status: Abandoned Application

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Steemers, Frank, GUNDERSON, Kevin L.
• Application Number:
  US11/943,554
• Publication Number:
  US 20080242560A1
• Time in Patent Office: Days
• US Class Current: 506/26
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

The present invention relates to methods for generating an array of amplified nucleic acid sequences. The methods can utilize amplicons that form nucleic acid balls that can be arrayed on a solid support. The invention additionally provides methods for obtaining targeted nucleic acid sequences.

Claims

1. A method of making an array, comprising

(a) providing a genomic DNA sample comprising a plurality of annealed capture probes having different sequences that are complementary to different target regions of said genomic DNA sample;

(b) sequentially treating said annealed capture probes with nucleotide analogs comprising reversible blocking groups under a first polymerase extension condition and then treating said annealed capture probes with nucleotide analogs comprising second blocking groups under a second condition, thereby producing a modified probe set comprising reversible blocking groups on a first plurality of said annealed capture probes and second blocking groups on a second plurality of said annealed capture probes,

wherein said first polymerase extension condition has higher extension fidelity than said second polymerase extension condition;

(c) removing said reversible blocking groups from said modified probe set and then adding at least one nucleotide to deblocked probes of said modified probe set, thereby forming a plurality of different extension products comprising said target regions; and

(d) attaching said different extension products to an array.

2. The method of claim 1, wherein said target regions attached to said array consist essentially of transcribed genomic regions.

3. The method of claim 1, wherein said target regions attached to said array consist essentially of exons.

4. The method of claim 1, wherein said at least one nucleotide that is added to said deblocked probes comprises a secondary label.

5. The method of claim 4, further comprising a step of isolating said plurality of different extension products via said secondary label prior to attaching said different extension products to said array.

6. The method of claim 1, further comprising circularizing said different extension products to generate a plurality of circularized nucleic acid molecules.

7. The method of claim 6, further comprising amplifying said circularized nucleic acid molecules to generate amplicons, wherein each of said amplicons comprises multiple copies of said extension products.

8. The method of claim 7, wherein said amplicons are compacted prior to attachment to said array.

9. The method of claim 8, wherein said compacted amplicons have an average diameter selected from about 0.1 μ

m, about 0.2 μ

m, about 0.5 μ

m, about 1 μ

m, 2 μ

m, about 3 μ

m, about 4 μ

m and about 5 μ

m.

10. The method of claim 9, wherein said amplicons are opened after distribution on said array.

11. A method of sequencing different target regions of a genomic DNA sample comprising making an array according to the method of claim 1 and sequencing one or more of said extension products attached to said array.

12. The method of claim 11, wherein said sequencing comprises a method selected from the group consisting of sequencing by synthesis, sequencing by ligation and sequencing by hybridization.

13. The method of claim 1, wherein said genomic DNA sample comprises an amplified product of genomic DNA comprising an exogenous bases and the method further comprises a step of cleaving said genomic DNA comprising said exogenous bases prior to step (d).

14. The method of claim 1, wherein said at least one nucleotide that is added to said deblocked probes comprises a nuclease resistant nucleotide and the method further comprises a step of cleaving said genomic DNA with said nuclease prior to step (d).

15. The method of claim 1, wherein said second condition comprises treatment with terminal deoxynucleotide transferase to produce said second blocking groups on said second plurality of said annealed capture probes.